Method for purifying an enzyme, a purified enzyme produced thereby, and use of this enzyme

ABSTRACT

The invention relates to a method for purifying at least one enzyme obtained in an excess fermentation of  Clostridium histolyticum.    
     It is provided for that the enzymes of the excess fermentation are separated by a multistage chromatography method by exclusively using chromatography materials on styrene/divinyl-benzene base and/or on base of in particular ceramic hydroxylapatite.

FIELD OF APPLICATION

[0001] The invention relates to a method for purifying at least one enzyme contained in an excess fermentation of Clostridium histolyticum and an enzyme produced according to this method as well as the use thereof.

PRIOR ART

[0002] The bacterium Clostridium histolyticum constitutes, when cultivated in a peptone containing nutrient medium extracellularly a complex enzym mixture which contains collagenases, different proteolytic enzymes as well as low molecular weight constituents. Type I and type II collagenases (clostridiopeptidase A, EC 3.4.24.3) with molecular weights in the range of 65 to 125 kD and isolectric points between 5 and 6.5 have been described as main constituents (Bond, van Wart; Biochemistry, 1984, 23, 3077-3085). Further main constituents are the SH-protease clostripain occuring as a heterodimer (clostridiopeptidase B, EC 3.4.22.8) with a molecular weight of approximately 59 kD and the bad characterized so-called neutral protease (caseinase) with a molecular weight of 34.5 kD determined by MALDI-TOF.

[0003] Collagenases are enzymes which cleave peptide bonds of the fiber proteine collagen. They are used in biochemistry and medicine, for example in order to isolate cells or cell bonds of tissues. Type I and type II collagenases differ in their activity with respect to high molecular collagen and small synthetic substrates. While type I collagenases preferably cleave high molecular collagen, type II collagenases react mainly with synthetic substrates such as, for example Pz-Pro-Leu-Gly-Pro-D-Arg (Wünsch, Heidrich; Z. Physiol. Chem. 333, 1963, 149-151), His-Pro (Nordwig, Wünsch; Z. Physiol. Chem. 316, 1959, 287) or Phe-Ala-Leu-Gly-Pro-Ala (van Wart, Steinbrink; Anal. Giochem. 113, 1981, 356-365). Both collagenase types can also be unequivocally differentiated by reversed phase chromatography (RPC).

[0004] The U.S. Pat. No. 5,332,503 describes a method for the chromatographic purification of collagenase of Clostridium histolyticum. This chromatography method comprises among other a gel filtration step as well as a dyestuff/ligand affinity chromatography by using reactive red agarose gel. The method shows decisive disadvantages to produce collagenases for pharmaceutical purposes under GMP conform conditions. So, the reactive red agarose gel used in the method involves the risk of a so-called bleeding of the chromatography material and of related toxicological problems. Furthermore, gel filtration steps are fundamentally time consuming and expensive and offer less efficient cleaning-in-place (CIP) possibilities. This make the use of this method difficult at a commercial scale. Moreover, the method requires the use of detergents so that high expenses are necessary for the purification validation and undesired changes of the end product can be caused. Finally, the method does not allow a separation of the type I and type II collagenases.

[0005] From the documents U.S. Pat. No. 5,830,741 and U.S. Pat. No. 5,952,215, we know a purification method for the separation of type I and type II collagenases which comprises a dyestuff/ligand affinity chromatography, a cation-exchange chromatography and an anion-exchange chromatography. Here also, there is the risk of bleeding of the dyestuff used in the affinity chromatography. Furthermore, the chromatography materials used allow only relatively low flow rates. Moreover all the chromatographic steps take place by gradient elution, whereby the enzymes bound to the chromagrography materials are eluted by linear salt concentration and/or pH gradients. As for the result, the known method is thus extremely time consuming.

[0006] Aim, Solution, Advantage

[0007] Against this background, the aim of this invention is to make available a method for separating and purifying at least one extracellular main enzym of Clostridium histolyticum which overcomes the described advantages of the prior art.

[0008] This aim is achieved by a method with the characteristics mentioned in claim 1. A very quick and cost-saving enzyme purification can be realized in that the enzymes of the excess fermentation of Clostridium histolyticum are separated by a single-stage or preferably a multiple-stage chromatography method by exclusively using chromatography materials on styrene/divinylbenzene base and/or on the base of ceramic hydroxylapatite, whereby high purity enzymes are obtained which are appropriate in particular for the use in pharmacy and/or biochemistry because of the absence of toxicologically dangerous substances. This method does not need any product contacting steps, what makes its use in the pharmaceutical field particularly advantageous. It can be used for the production of sterile collagenase enzymes. This being, by styrene/divinyl benzene, a copolymer is understood which consists of polystyrene cross-linked with divinyl benzene. Through the substitution of this base matrix with the most different functional groups, a bond of proteins to the material and thus a separation thereof is made possible. Depending on the choice of the functional group, different bond and separating mechanisms, for example cation-exchange or ion-exchange mechanisms, can be used. On the other hand, with hydroxylapatite the matter is of a phosphate of calcium of the total formula Ca₁₀(PO₄)₆(OH)₂ which allows a protein bond because of ionic electrostatic interactions as well as a stronger ionic complex bond. However, in detail the bond mechanism of proteins to hydroxylapatite has not been yet understood.

[0009] The chromatography materials used allow the adjusting of high flow rates with good separating properties so that there result very short purification times. This is particularly valid if, according to a preferred embodiment of the invention, sintered, thus ceramic hydroylapatite is used. The use of ceramic hydroxylapatite has the advantage, compared to non-sintered (cristalline) hydroxylapatite, that the material can be produced reproduceably and because of its porous structure only very low back pressures are generated so that the risk of column damages is minimized and high linear flow rates are made possible. The elution from sintered hydroxylapatite material is carried out preferably at flow rates of at least 200 cm/h, in particular of at least 300 cm/h. In the case of the polymere styrene/divinylbenzene material, flow rates of at least 500 cm/h are even preferred, in particular of at least 1000 cm/h. In this way, the processing times can be considerably reduced compared to known methods.

[0010] A further processing time reduction as well as a simplification of the process expenditure is achieved in that at least one chromatography stage, preferably all chromatography stages, is carried out as a stepwise elution. Compared with a gradient elution, elution means and thus expensive puffer substances can thus be saved. Moreover, the generation of a gradient is a problem on the process scale.

[0011] According to an advantageous configuration of the invention, the chromatography materials are selected in such a way that the individual chromatographic steps are based exclusively on electrostatic interactions and/or on ion bond and/or ion complex bond. Particularly advantageously, the method comprises at least one anion-exchange chromatic stage and/or at least one cation-exchange chromatic stage and/or at least one hydroxylapatite chromatic stage. Particularly good results have been obtained in a three-stage chromatography method, whereby a first stage is carried out on sintered hydroxylapatite material, a second stage on an anion-exchange material on styrene/divinylbenzene base and a third chromatographic stage on a cation-exchange material on styrene/divinylbenzene base, preferably in said order; however, other orders are also possible; the number of stages can vary as well.

[0012] Preferably, the method according to the invention thus does not comprise any time consuming and cost intensive gel filtration steps so that the risk of a self-digesting of enzymes to be separated is minimized. Furthermore, the abandonment of gel filtrations makes possible the carrying out of the chromatographic stages in a wide temperature range between 4 and 25° C., thus also at ambient temperature. Moreover, the method does not comprise any affinity chromatographic stages so that the risk of bleeding, thus of washing out of the affinity ligands of the chromatography material does not exist. Finally, the method according to the invention does not provide any protein precipitation steps which can cause undesired protein structure changes. Furthermore, thus there results the advantage that no detergents or chaotropic substances (ammonium sulphate, polyethyleneglycol etc.) are used, the subsequent removing of which is always bound to a high process expenditure.

[0013] The chromatography materials used have the further advantage to be chemically inert to a wide extent and to be able to be purified with relatively highly concentrated alkaline lyes, for example with trimolar soda lye. This guarantees a very effective purification and thus a functional maintenance of the materials as well as a good reproducibility of the method. The high pressure stability of the chromatograhy materials further allows the use in high pressure liquid chromatography methods (HPCL). Because of the absence of toxicologically dangerous, process induced substances such as for example affinity ligands or detergents, the proteins purified according to the invention, in particular type I and/or type II collagenase and/or clostripain and/or neutral protease (caseinase), can be used particularly advantageously for pharmaceutical or biochemical purposes.

[0014] Further preferred configurations of the invention result from the other characteristics indicated in the subclaims.

[0015] Furthermore, the invention relates to an enzyme purified according to the method according to the claims 31 and 31 as well as the use of an enzyme produced according to the method according to claim 32.

SHORT DESCRIPTION OF THE DRAWING

[0016] The invention will be explained by way of example with the aid of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION AND BEST WAY OF CARRYING OUT THE INVENTION

[0017] After the cultivation of Clostridium histolyticum has taken place in a fermentation medium of animal or vegetable origin, the cells and other non soluble constituents are separated from the excess fermentation, for example by centrifugation or filtration. The excess fermentation which contains type I and type II collagenases, clostripain and caseinase as main constituents can be upgraded in the usual way before the chromatographic separation of these proteins.

[0018] The excess fermentation is pumped in a first step of the method onto a chromatography column filled with ceramic hydroxylapatite material of type I or type II (CHT), whereby said enzymes bind with the hydroxylapatite besides different other components. At temperatures of 4 to 25° C. and a pH-value of 6 to 9, the elution takes place as a stepwise elution with linear flow rates >300 cm/h. This being, a phosphate buffer which contains 0 to 1000 mM of an alkali halide is convenient, whereby the phosphate concentration is gradually increased from 10 to 350 mM phosphate. Alternatively or additionally, the pH value of the buffer can be gradually increased from 6 to 9. Preferably three elution stages are carried out. The fraction 1 obtained in the first elution stage contains exclusively low molecular constituents. The fraction 2 of the second elution stage contains besides low molecular components neutral protease (caseinase). The fraction 3 of the third elution stage also contains low molecular components, the whole clostripain as well as all the type I and type II collagenases.

[0019] The fraction 3 will be desalted for example by ultrafiltration/diafiltration or nanofiltration or dialysis and eventually upgraded. Then, the desalted solution will be submitted to an anion-exchange chromatography in a second chromatographic step. A chromatography material on styrene/divinylbenzene base will be used for this and will be functionalized for example with a quaternary ammonium group. This being, commercially available materials can be used (for example Source of the Pharmacia company, POROS of the PerSeptive company, Makroprep of the Biorad company). A separation of the cationic or nonionic low molecular constituents takes place already when loading the anion exchanger with the fraction 3. The elution of the bound constituents takes place at 4 to 25° C. in a buffered system in the pH range of 7 to 9.5 by stepwise elution with a linear flow rate above 500 cm/h, whereby an alkali halide or alkali-alkaline earth halide concentration gradually varies from 1 to 1000 mM and/or the pH value from 9.5 to 6. Preferably two fractions are separated, whereby the fraction 4 of the first elution stage contains clostripain and type II collagenase and the fraction 5 of the second elution stage also contains clostripain as well as type I collagenase.

[0020] The fractions 4 and 5 are submitted to a cation-exchange chromatography in a third chromatographic stage separately from each other. This being, a colum filling basing on styrene/divinylbenzene material is also used which is however functionalized here with a cation binding group, for example SO₃H. The above mentioned commercial materials can also be used here. The elution of the cation-exchanger will be carried out at 4 to 25° C. in a buffered system in the pH range of 5.7 to 7 for linear flow rates of at least 500 cm/h with a stepweise elution. This being, an alkali halide or alikali-alkaline earth halide concentration between 0 and 300 mM is gradually adjusted in the elution buffer and/or the pH value between 5 and 7. The elution is preferably carried out in two stages, whereby the respective collagenase is obtained in the first elution stage and clostripain in the second elution stage.

[0021] Embodiment

[0022] A culture of Clostridium histolyticum is fermented by using an animal or vegetable nutrient medium in liquid culture according to standard methods up to a desired cell density. After separation of the cells with usual methods, for example by centrifugation or filtration, 2000 ml of the concentrated excess fermentation are pumped with a linear flow rate of 300 cm/h onto a chromatography column filled with 1700 ml type I ceramic hydroxylapatite. The components bound to the hydroxylapatite column are eluted at 20 to 25° C. with a linear flow rate of 300 cm/h in three stages with phosphate buffer, whereby the phosphate concentration is gradually increased. In the first elution stage, the elution takes place with approximately 10 CV (column volumes) with 10 mM phosphate buffer/100 mM NaCl, whereby the fraction 1 is obtained which contains mainly low molecular constituents. Then 60 mM phosphate buffer/100 mM NaCl are eluted with approximately 3 CV and the fraction 2 is obtained which contains caseinase and also low molecular components. For the third elution stage, 200 mM phosphate buffer/100 mM NaCl are eluted with approximately 5 CV. The fraction 3 collected in this way contains the enzymes clostripain and all the type I and type II collagenases. The fraction 2 is desalted and lyophilized. A separation of the low molecular constituents of caseinase can eventually be achieved with standard methods.

[0023] The fraction 3 is desalted by ultrafiltration/diafiltration or nanofiltration or dialysis and eventually upgraded. Then the fraction 3 is adjusted to a pH value of 9.0-9.3 with an appropriate buffer, for example 500 mM Tris pH 9.0-9.3. The buffered solution is pumped at 20-25° C. with a linear flow rate of approximately 1000 cm/h over a styrene/divinylbenzene column (POROS 50 Pl of the PerSeptive company) functionalized as an anion-exchanger. The column is then washed with approximately 5 CV tris-buffer. The elution takes place at 20-25° C. and a linear flow rate of approximately 1000 cm/h in two elution stages with an increasing salt concentration. The fraction 4 is obtained by elution with 40 mM tris-buffer/6 mM CaCl₂/30 mM NaCl pH 9.0-9.3 and contains the enzymes clostripain as well as type II collagenase. The fraction 5 which also contains clostripain as well as type I collagenase is obtained in the second elution stage by elution with 40 mM tris/6 mM CaCl₂/70 mM NaCl pH 9.0-9.3.

[0024] The fractions 4 and 5 are desalted for example by dialysis for 24 hours against 50 l H₂O and adjusted with 50 mM MES buffer to a pH value of 5.9-6.1. Both fractions are chromatographed separately the one from the other on a cation-exchanger column on styrene/divinylbenzene material (for example POROS HS of the PerSeptive company). For this purpose, the fractions are loaded with a linear flow rate of approximately 700 cm/h at 20-25° C. onto the column and eluted under the same conditions. The elution of type II collagenase from fraction 4 or of type I collagenase from fraction 5 takes place respectively in a first elution stage with 10 mM MES buffer/20-40 mM NaCl pH 5.9-6.1 at 20-25° C. The clostripain containing solutions will be combined. All the solutions are desalted, dialyzed against 2 mM CaAc₂ and then lyophilized.

[0025] A determination of the respective enzyme activities of the obtained enzymes took place according to known methods (see table). Furthermore, the purity of the type I collagenase, type II collagenase enzymes and clostripain has been determined by reversed phase chromatography. The results are summarized in the table below: TABLE Activity Purity^(e) Type II collagenase 18100 U/g^(a) Approx. 82% Type I collagenase 5180 U/g^(b) Approx. 85% Clostripain 322 U/mg^(c) Approx. 90% Neutral protease 1560 U/mg^(d) 

1. A method for the partial or complete purification at least of one enzyme contained in an excess fermentation of Clostridium histolyticum, characterized in that the enzymes of the excess fermentation are separated by a chromatography method by exclusively using chromatography materials on styrene/divinyl-benzene base and/or on base of in particular ceramic hydroxylapatite.
 2. A method according to claim 1, characterized in that the chromatography method is carried out in a single stage or preferable in several stages.
 3. A method according to claim 1 or 2, characterized in that the enzyme is type I collaganease and/or type II collagenase and/or clostripain and/or neutral protease (caseinase).
 4. A method according to any of the preceding claims, characterized in that sintered hydroxylapatite is used.
 5. A method according to any of the preceding claims, characterized in that the chromatography method comprises exclusively steps which are based on electrostatic interactions and/or on ion bond and/or ion complex bond.
 6. A method according to claim 5, characterized in that the chromatography method comprises at least one anion-exchange chromatic stage and/or at least one cation-exchange chromatic stage and/or at least one hydroxylapatite chromatic stage.
 7. A method according to any of the preceding claims, characterized in that at least one chromatography stage, in particular all chromatography stages, is carried out as a stepwise elution.
 8. A method according to any of the preceding claims, characterized in that the chromatography method is a two-stage or three-stage method.
 9. A method according to claim 8, characterized in that the chromatography method comprises a first chromatographic stage on sintered hydroxylapatite material, a second chromatographic stage on an anion-exchanger material on styrene/divinylbenzene base and eventually a third chromatographic stage on a cation-exchanger material on styrene/divinylbenzene base.
 10. A method according to any of the claims 8 or 9, characterized in that the first chromatographic stage is carried out in at least two elution stages, whereby type I neutral protease (caseinase) and type II collagenase and clostripain is separated.
 11. A method according to any of the claims 8 to 10, characterized in that a fraction substantially freed from caseinase from the first chromatographic stage is submitted to the second chromatographic stage with at least two elution stages, whereby type I collagenase and type II collagenase are separated from each other.
 12. A method according to any of the claims 8 to 11, characterized in that a type I collagenase containing fraction from the second chromatrographic stage is submitted to the third chromatographic stage with at least two elution stages, whereby type I collagenase is separated from clostripain.
 13. A method according to any of the claims 9 to 12, characterized in that a type II collagenase containing fraction from the second chromatographic stage is submitted to the third chromatographic stage with at least two elution stages, whereby type II collagenase is separated from clostripain.
 14. A method according to any of the preceding claims, characterized in that the elution is carried out with linear flow rates.
 15. A method according to any of the preceding claims, characterized in that the elution is carried out from the sintered hydroxylapatite material at flow rates of at least 200 cm/h, in particular of at least 300 cm/h.
 16. A method according to any of the preceding claims, characterized in that the elution is carried out from the styrene/divinylbenzene material at flow rates of at least 500 cm/h, in particular of at least 1000 cm/h.
 17. A method according to any of the preceding claims, characterized in that the method does not comprise any gel filtration steps.
 18. A method according to any of the preceding claims, characterized in that the method does not comprise any protein precipitation steps.
 19. A method according to any of the preceding claims, characterized in that the method comprises degrading steps for low molecular substances, host cell proteins, endotoxins and/or nucleic acids, in particular DNA.
 20. A method according to any of the preceding claims, characterized in that the chromatographic stages are carried out at temperatures between 4 and 25° C.
 21. A method according to any of the preceding claims, characterized in that certified chromatography media are used.
 22. A method according to any of the preceding claims, characterized in that pressure stable chromatography materials are used.
 23. A method according to any of the preceding claims, characterized in that a purification of the chromatography materials is carried out with a concentrated alkaline lye, in particular with three molar soda lye.
 24. A method according to any of the preceding claims, characterized in that the chromatography stages are carried out as column chromatography.
 25. A method according to any of the preceding claims, characterized in that type II collagenase with a specific activity of at least 13000 U/g, in particular of at least 18000 U/g, is obtained.
 26. A method according to any of the preceding claims, characterized in that type I collagenase with a specific activity of at least 3000 U/mg, in particular of at least 5000 U/mg, is obtained.
 27. A method according to any of the preceding claims, characterized in that clostripain with a specific activity of at least 200 U/mg, in particular of at least 300 U/mg, is obtained.
 28. A method according to any of the preceding claims, characterized in that neutral protease (caseinase) with a specific activity of at least 1200 U/mg, in particular of at least 1500 U/mg, is obtained.
 29. A method according to any of the preceding claims, characterized in that type II collagenase and/or type I collagenase and/or clostripain respectively with a purity of at least 70%, in particular of at least 80%, are obtained.
 30. Partially or completely purified enzyme contained in an excess fermentation of Clostridium histolyticum, such as type I collagenase and/or type II collagenase and/or clostripain and/or neutral protease (caseinase), characterized in that the enzymes of the excess fermentation are separated by a chromatography method by exclusively using chromatography materials on styrene/divinyl/divinyl-benzene base and/or on base of in particular ceramic hydroxylapatite.
 31. Enzyme, characterized in that the enzyme is produced according to a method according to claim 1 to
 30. 32. Use of an enzyme purified according to a method according to claim 1 to 29 for pharmaceutical and/or biochemical purposes. 